ABSTRACT Our work to date has provided new important insights into the early hematopoietic hierarchy. These studies have also highlighted a key role for Ikaros and its associate the chromatin remodeler Mi-2[unreadable] in fate decisions made by hematopoietic stem cells and multipotent progenitors. We now continue with our efforts to delineate the genetic and epigenetic mechanisms that promote the multi-lineage state and provide lineage restrictions. In Aim 1, we study the activation (priming) of cell type-specific transcriptional programs in the hematopoietic stem cell and its early progeny. We investigate the dependence of these programs on Ikaros and Mi-2[unreadable]. Questions addressed here relate to where transcriptional priming first occurs, how many lineages are involved, how they are subsequently resolved and how this molecular process is influenced by Ikaros and Mi-2[unreadable]. In Aim 2, we evaluate the functional interactions between Mi-2[unreadable] and Ikaros in lineage restriction using a series of genetic and molecular approaches. Mouse genetic models are utilized to test the effects of overt Mi- 2[unreadable] deletion in lymphoid lineage restriction either alone or in combination with Ikaros. The biological implications of loss or gain of select Ikaros and Mi-2[unreadable] targets from early-primed lineage signatures as potential downstream regulators of the differentiation process are tested. In Aim 3, we study the molecular mechanisms by which Ikaros and Mi-2[unreadable] control lineage priming in early progenitors. Targets of Ikaros and Mi-2[unreadable] are pursued using both genome-wide and candidate-gene biochemical approaches. Ikaros and Mi-2[unreadable] gene targets and their chromatin environments that support their early priming are pursued here. Finally, a cross validation of databases with Ikaros and Mi-2[unreadable] gene targets provided by the expression and chromatin studies is undertaken to delineate the genetic hierarchy in control of early hematopoiesis. The proposed studies integrate state-of-the-art approaches in cellular and molecular biology, biochemistry, genetics and computational sciences to investigate key questions in early hematopoiesis. The focus on how cell fate decisions are generated through the implementation of genetic networks and their associated epigenetic regulators in the HSC and its early progeny is likely to provide new important and farreaching paradigms that may impact both normal and cancer development. Implementation of regulatory circuits deduced from these studies may allow us to generate clinically useful protocols to manipulate HSC and progenitor cells in a highly controlled fashion.